IKK-alpha proteins, nucleic acids and methods

ABSTRACT

The invention provides methods and compositions relating to an IκB kinase, IKK-α, and related nucleic acids. The polypeptides may be produced recombinantly from transformed host cells from the disclosed IKK-α encoding nucleic acids or purified from human cells. The invention provides isolated IKK-α hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-α genes, IKK-α-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35USC120 to U.S. Ser. No.09/109,986, filed on Jul. 2, 1998, now allowed, which claims priority toU.S. Ser. No. 08/890,854, filed Jul. 10, 1997, now U.S. Pat. No.6,235,512, which claims priority to U.S. Ser. No. 08/887,115 filed Jul.1, 1997, abandoned, all of which are incorporated herein by reference.

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of this invention is proteins involved in transcriptionfactor activation.

[0004] 2. Background

[0005] Cytokines trigger changes in gene expression by modifying theactivity of otherwise latent transcription factors (Hill and Treisman,1995). Nuclear factor κB (NF-κB) is a prominent example of how such anexternal stimulus is converted into an active transcription factor(Verma et al., 1995). The NF-κB system is composed of homo- andheterodimers of members of the Rel family of related transcriptionfactors that control the expression of numerous immune and inflammatoryresponse genes as well as important viral genes (Lenardo and Baltimore,1989; Baeuerle and Henkel, 1994). The activity of NF-κB transcriptionfactors is regulated by their subcellular localization (Verma et al.,1995). In most cell types, NF-κB is present as a heterodimer comprisingof a 50 kDa and a 65 kDa subunit. This heterodimer is sequestered in thecytoplasm in association with IκBα a member of the IκB family ofinhibitory proteins (Finco and Baldwin, 1995; Thanos and Maniatis, 1995;Verma et al., 1995). IκBα masks the nuclear localization signal of NF-κBand thereby prevents NF-κB nuclear translocation. Conversion of NF-κBinto an active transcription factor that translocates into the nucleusand binds to cognate DNA sequences requires the phosphorylation andsubsequent ubiquitin-dependent degradation of IκBα in the 26sproteasome. Signal-induced phosphorylation of IκBα occurs at serines 32and 36. Mutation of one or both of these serines renders IκBα resistantto ubiquitination and proteolytic degradation (Chen et al., 1995).

[0006] The pleiotropic cytokines tumor necrosis factor (TNF) andinterleukin-1 (IL-1) are among the physiological inducers of IκBphosphorylation and subsequent NF-κB activation (Osborn et al., 1989;Beg et al., 1993). Although TNF and IL-1 initiate signaling cascadesleading to NF-κB activation via distinct families of cell-surfacereceptors (Smith et al., 1994; Dinarello, 1996), both pathways utilizemembers of the TNF receptor-associated factor (TRAF) family of adaptorproteins as signal transducers (Rothe et al., 1995; Hsu et al., 1996;Cao et al., 1996b). TRAF proteins were originally found to associatedirectly with the cytoplasmic domains of several members of the TNFreceptor family including the 75 kDa TNF receptor (TNFR2), CD40, CD30,and the lymphotoxin-β receptor (Rothe et al., 1994; Hu et al., 1994;Cheng et al., 1995; Mosialos et al., 1995; Song and Donner, 1995; Satoet al., 1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al.,1996). In addition, TRAF proteins are recruited indirectly to the 55 kDaTNF receptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996).Activation of NF-κB by TNF requires TRAF2 (Rothe et al., 1995; Hsu etal., 1996). TRAF5 has also been implicated in NF-κB activation bymembers of the TNF receptor family (Nakano et al., 1996). In contrast,TRAF6 participates in NF-κB activation by IL-I (Cao et al., 1996b). UponIL-1 treatment, TRAF6 associates with IRAK, a serine-threonine kinasethat binds to the IL-1 receptor complex (Cao et al., 1996a).

[0007] The NF-κB-inducing kinase (NIK) is a member of the MAP kinasekinase kinase (MAP3K) family that was identified as a TRAF2-interactingprotein (Malinin et al., 1997). NIK activates NF-κB when overexpressed,and kinase-inactive mutants of NIK comprising its TRAF2-interactingC-terminal domain (NIK₍₆₂₄₋₉₄₇₎) or lacking two crucial lysine residuesin its kinase domain (NIK_((KK429-430AA))) behave as dominant-negativeinhibitors that suppress TNF-, IL-1-, and TRAF2-induced NF-κB activation(Malinin et al., 1997). Recently, NIK was found to associate withadditional members of the TRAF family, including TRAF5 and TRAF6.Catalytically inactive mutants of NIK also inhibited TRAF5- andTRAF6-induced NF-κB activation, thus providing a unifying concept forNIK as a common mediator in the NF-κB signaling cascades triggered byTNF and IL-1 downstream of TRAFs.

[0008] Here, we disclose a novel human kinase IκB Kinase, IKK-α, as aNIK-interacting protein. IKK-α has sequence similarity to the conceptualtranslate of a previously identified open reading frame (SEQ ID NO:5)postulated to encode a serine-threonine kinase of unknown function(‘Conserved Helix-loop-helix Ubiquitous Kinase’ or CHUK, Connelly andMarcu, 1995; Mock et al., 1995). Catalytically inactive mutants of TKK-αare shown to suppress NF-κB activation induced by TNF and IL-1stimulation as well as by TRAF and NIK overexpression; transientlyexpressed IKK-α is shown to associate with the endogenous IκBα complex;and IKK-α is shown to phosphorylate IκBα on serines 32 and 36.

SUMMARY OF THE INVENTION

[0009] The invention provides methods and compositions relating toisolated IKK-α polypeptides, related nucleic acids, polypeptide domainsthereof having IKK-α-specific structure and activity and modulators ofIKK-α function, particularly IκB kinase activity. IKK-α polypeptides canregulate NFκB activation and hence provide important regulators of cellfunction. The polypeptides may be produced recombinantly fromtransformed host cells from the subject IKK-α polypeptide encodingnucleic acids or purified from mammalian cells. The invention providesisolated IKK-α hybridization probes and primers capable of specificallyhybridizing with the disclosed IKK-α gene, IKK-α-specific binding agentssuch as specific antibodies, and methods of making and using the subjectcompositions in diagnosis (e.g. genetic hybridization screens for IKK-αtranscripts), therapy (e.g. IKK-α kinase inhibitors to inhibit TNFsignal transduction) and in the biopharmaceutical industry (e.g. asimmunogens, reagents for isolating other transcriptional regulators,reagents for screening chemical libraries for lead pharmacologicalagents, etc.).

DETAILED DESCRIPTION OF THE INVENTION

[0010] The nucleotide sequence of a natural cDNA encoding a human IKK-αpolypeptide is shown as SEQ ID NO:3, and the full conceptual translateis shown as SEQ ID NO:4. The IKK-α polypeptides of the invention includeincomplete translates of SEQ ID NO:3, particularly of SEQ ID NO:3,residues 1-638, which translates and deletion mutants of SEQ ID NO:4have human IKK-α-specific amino acid sequence, binding specificity orfunction and comprise at least one of Cys30, Leu403, Glu543, Leu604,Thr679, Ser680, Pro684, Thr686 and Ser687. Preferred translates/deletionmutants comprise at least a 6, preferably at least a 12, more preferablyat least an 18 residue Cys30, Leu403, Glu543, Leu604, Thr679, Ser680,Pro684, Thr686 or Ser687.-containing domain of SEQ ID NO:4, preferablyincluding at least 8, more preferably at least 12, most preferably atleast 20 contiguous residues which immediately flank said residue onone, preferably both sides, with said residue preferably residing withinsaid contigous residues, see, e.g. Table 1A; which mutants providehIKK-α specific epitopes and immunogens. TABLE 1A Exemplary IKK-αpolypeptides having IKK-α binding specificity αΔ1 (SEQ ID NO: 4,residues 1-30) αΔ2 (SEQ ID NO: 4, residues 22-31) αΔ3 (SEQ ID NO: 4,residues 599-608) αΔ4 (SEQ ID NO: 4, residues 601-681) αΔ5 (SEQ ID NO:4, residues 604-679) αΔ6 (SEQ ID NO: 4, residues 670-687) αΔ7 (SEQ IDNO: 4, residues 679-687) αΔ8 (SEQ ID NO: 4, residues 680-690) αΔ9 (SEQID NO: 4, residues 684-695) αΔ10 (SEQ ID NO: 4, residues 686-699) αΔ11(SEQ ID NO: 4, residues 312-345) αΔ12 (SEQ ID NO: 4, residues 419-444)αΔ13 (SEQ ID NO: 4, residues 495-503) αΔ14 (SEQ ID NO: 4, residues565-590) αΔ15 (SEQ ID NO: 4, residues 610-627) αΔ16 (SEQ ID NO: 4,residues 627-638) αΔ17 (SEQ ID NO: 4, residues 715-740) αΔ18 (SEQ ID NO:4, residues 737-745)

[0011] In a particular embodiment, the invention provides IKK-αGlu⁵⁴³polypeptides, IKK-αGlu⁵⁴³ polypeptide-encoding nucleicacids/polynucleotides, and IKK-αGlu⁵⁴³ polypeptide-based methods(below), which IKK-αGlu⁵⁴³ polypeptides comprise at least 8, preferablyat least 10, more preferably at least 12, more preferably at least 16,most preferably at least 24 consecutive amino acid residues of the aminoacid sequence set forth as SEQ ID NO:4, which consecutive amino acidresidues comprise the amino acid residue 543 (Glu) of SEQ ID NO:4.Exemplary IKK-αGlu⁵⁴³ polypeptides having IKK-αGlu⁵⁴³ bindingspecificity and immunologically distinguishable from IKK-αGly⁵⁴³ areshown in Table 1B. TABLE 1B Exemplary IKK-αGlu⁵⁴³ polypeptides havingIKK-αGlu⁵⁴³ binding specificity αΔ19 (SEQ ID NO: 4, residues 540-548)αΔ20 (SEQ ID NO: 4, residues 543-550) αΔ21 (SEQ ID NO: 4, residues536-543) αΔ22 (SEQ ID NO: 4, residues 534-554) αΔ23 (SEQ ID NO: 4,residues 533-543) αΔ24 (SEQ ID NO: 4, residues 543-563) αΔ25 (SEQ ID NO:4, residues 542-549) αΔ26 (SEQ ID NO: 4, residues 538-545) αΔ27 (SEQ IDNO: 4, residues 541-547) αΔ28 (SEQ ID NO: 4, residues 403-543) αΔ29 (SEQID NO: 4, residues 403-604) αΔ30 (SEQ ID NO: 4, residues 403-679) αΔ31(SEQ ID NO: 4, residues 403-680) αΔ32 (SEQ ID NO: 4, residues 403-687)αΔ33 (SEQ ID NO: 4, residues 543-604) αΔ34 (SEQ ID NO: 4, residues543-679) αΔ35 (SEQ ID NO: 4, residues 543-684) αΔ36 (SEQ ID NO: 4,residues 543-687)

[0012] The subject domains provide IKK-α domain specific activity orfunction, such as IKK-α-specific kinase or kinase inhibitory activity,NIK-binding or binding inhibitory activity, IκB-binding or bindinginhibitory activity, NFκB activating or inhibitory activity or antibodybinding. Preferred domains phosphorylate at least one and preferablyboth the serine 32 and 36 of IκB (Verma, I. M., et al. (1995)). As usedherein, Ser32 and Ser36 of IκB refers collectively to the two serineresidues which are part of the consensus sequence DSGL/IXSM/L (e.g. ser32 and 36 in IκBα, ser 19 and 23 in IκBβ, and ser 157 and 161, or 18 and22, depending on the usage of methionines, in IκBε, respectively.

[0013] IKK-α-specific activity or function may be determined byconvenient in vitro, cell-based, or in vivo assays: e.g. in vitrobinding assays, cell culture assays, in animals (e.g. gene therapy,transgenics, etc.), etc. Binding assays encompass any assay where themolecular interaction of an IKK-α polypeptide with a binding target isevaluated. The binding target may be a natural intracellular bindingtarget such as an IKK-α substrate, a IKK-α regulating protein or otherregulator that directly modulates IKK-α activity or its localization; ornon-natural binding target such a specific immune protein such as anantibody, or an IKK-α specific agent such as those identified inscreening assays such as described below. IKK-α-binding specificity mayassayed by kinase activity or binding equilibrium constants (usually atleast about 10⁷ M⁻¹, preferably at least about 10⁸ M⁻¹, more preferablyat least about 10⁹ M⁻¹), by the ability of the subject polypeptide tofunction as negative mutants in IKK-α-expressing cells, to elicit IKK-αspecific antibody in a heterologous host (e.g a rodent or rabbit), etc.In any event, the IKK-α binding specificity of the subject IKK-αpolypeptides necessarily distinguishes the murine and human CHUKsequences of Connelly and Marcu (1995) as well as IKK-β (SEQ ID NO:4).

[0014] The claimed IKK-α polypeptides are isolated or pure: an“isolated” polypeptide is unaccompanied by at least some of the materialwith which it is associated in its natural state, preferablyconstituting at least about 0.5%, and more preferably at least about 5%by weight of the total polypeptide in a given sample and a purepolypeptide constitutes at least about 90%, and preferably at leastabout 99% by weight of the total polypeptide in a given sample. In aparticular embodiments, IKK-α polypeptides are isolated from a MKP-1precipitable complex, isolated from a IKK complex, and/or isolated fromIKK-β. The IKK-α polypeptides and polypeptide domains may besynthesized, produced by recombinant technology, or purified frommammalian, preferably human cells. A wide variety of molecular andbiochemical methods are available for biochemical synthesis, molecularexpression and purification of the subject compositions, see e.g.Molecular Cloning, A Laboratory Manual (Sambrook, et al Cold SpringHarbor Laboratory), Current Protocols in Molecular Biology (Eds.Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or thatare otherwise known in the art.

[0015] The invention provides binding agents specific to IKKpolypeptides, preferably the claimed IKK-α polypeptides, includingsubstrates, agonists, antagonists, natural intracellular bindingtargets, etc., methods of identifying and making such agents, and theiruse in diagnosis, therapy and pharmaceutical development. For example,specific binding agents are useful in a variety of diagnostic andtherapeutic applications, especially where disease or disease prognosisis associated with improper utilization of a pathway involving thesubject proteins, e.g. NF-κB activation. Novel IKK-specific bindingagents include IKK-specific receptors, such as somatically recombinedpolypeptide receptors like specific antibodies or T-cell antigenreceptors (see, e.g Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory) and other natural intracellularbinding agents identified with assays such as one-, two- andthree-hybrid screens, non-natural intracellular binding agentsidentified in screens of chemical libraries such as described below,etc. Agents of particular interest modulate IKK function, e.g.IKK-dependent transcriptional activation. For example, a wide variety ofinhibitors of IKK IκB kinase activity may be used to regulate signaltransduction involving IκB. Exemplary IKK IκB kinase inhibitors includeknown classes of serine/threonine kinase (e.g. PKC) inhibitors such ascompetitive inhibitors of ATP and substrate binding, antibiotics,IKK-derived peptide inhibitors, etc., see Tables II and III. IKKspecificity and activity are readily quantified in high throughputkinase assays using panels of protein kinases (see cited references andExamples).

[0016] Preferred inhibitors include natural compounds such asstaurosporine (Omura S, et al. J Antibiot (Tokyo) July1995;48(7):535-48), produced by a marine organism, and syntheticcompounds such as PD 153035, which also potently inhibits the EGFreceptor protein kinase (Fry D W et al. Science Aug. 19,1994;265(5175):1093-5). Members of the tyrphostin family of syntheticprotein kinase inhibitors are also useful; these include compounds whichare pure ATP competitors, compounds which are pure substratecompetitors, and compounds which are mixed competitors: compete withboth ATP and substrate (Levitzki A and Gazit A, Science Mar. 24,1995;267(5205):1782-8). Additional IKK inhibitors include peptide-basedsubstrate competitors endogenously made by the mammalian cell, e.g. PKI(protein kinase inhibitor, Seasholtz A F et al., Proc Natl Acad Sci USAFeb. 28, 1995;92(5):1734-8), or proteins inhibiting cdc kinases(Correa-Bordes J and Nurse P, Cell 1995 Dec 15;83(6):1001-9). Additionalsmall peptide based substrate competitive kinase inhibitors andallosteric inhibitors (inhibitory mechanisms independent of ATP orsubstrate competition) are readily generated by established methods(Hvalby O, et al. Proc Natl Acad Sci USA May 24, 1994;91(11):4761-5;Barja P, et al., Cell Immunol January 1994;153(1):28-38; Villar-PalasiC, Biochim Biophys Acta Dec. 30, 1994;1224(3):384-8; Liu W Z, et al.,Biochemistry Aug. 23, 1994;33(33):10120-6). TABLE II Selected SmallMolecule IKK Kinase Inhibitors HA-100¹ Iso-H7¹² A-3¹⁸ Chelerythrine² PKC19-31 HA1004^(19,20) Staurosporine^(3,4,5) H-7^(13,3,14) K-252a^(16,5)Calphostin C^(6,7,8,9) H-89¹⁵ KT5823¹⁶ K-252b¹⁰ KT5720¹⁶ ML-9²¹ PKC19-36¹¹ cAMP-depPKinhib¹⁷ KT5926²²

[0017] Citations

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[0022] 5. Tischler, A. S., et al. J. Neurochemistry 55: 1159 (1990)

[0023] 6. Bruns, R. F., et al. Biochem Biophys Res Corn 176: 288 (1991)

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[0025] 8. Tamaoki, T., et al Adv2nd Mass Phosphoprotein Res 24:497(1990)

[0026] 9. Tamaoki, T., et al. Biotechnology 8: 732 (1990)

[0027] 10. Yasuzawa, T. J. Antibiotics 39: 1972 (1986)

[0028] 11. House, C., et al. Science 238: 1726 (1987)

[0029] 12. Quick, J., et al. Biochem. Biophys. Res. Corn. 167: 657(1992)

[0030] 13. Bouli, N. M. and Davis, M. Brain Res. 525: 198 (1990)

[0031] 14. Takahashi, I., et al. J. Pharmacol. Exp. Ther. 255: 1218(1990)

[0032] 15. Chijiwa, T., et al. J. Biol. Chem. 265: 5267 (1990)

[0033] 16. Kase, H., et al. Biochem. Biophys. Res. Corn. 142: 436 (1987)

[0034] 17. Cheng, H. C., et al. J. Biol. Chem. 261: 989 (1986)

[0035] 18. Inagaki, M., et al. Mol. Pharmnacol. 29: 577 (1986)

[0036] 19. Asano, T. and Hidaka, H. J Pharmaco. Exp Ther 231:141 (1984)

[0037] 20. Hidaka, H., et al. Biochemistry 23: 5036 (1984)

[0038] 21. Nagatsu, T., et al. Biochem Biophys Res Corn 143:1045 (1987)

[0039] 22. Nakanishi, S., et al. Mol. Pharnacol. 37: 482 (1990) TABLEIII Selected Peptidyl IKK Kinase Inhibitors hIκBα, residues 24-39, 32AlahIKK-α, Δ5-203 hIκBα, residues 29-47, 36Ala hIKK-α, Δ1-178 hIκBα,residues 26-46, 32/36Ala hIKK-α, Δ368-756 hIκBβ, residues 25-38, 32AlahIKK-α, Δ460-748 hIκBβ, residues 30-41, 36Ala hIKK-α, Δ1-289 hIκBβ,residues 26-46, 32/36Ala hIKK-α, Δ12-219 hIκBε, residues 24-40, 32AlahIKK-α, Δ307-745 hIκBε, residues 31-50, 36Ala hIKK-α, Δ319-644 hIκBε,residues 27-44, 32/36Ala

[0040] Accordingly, the invention provides methods for modulating signaltransduction involving IκB in a cell comprising the step of modulatingIKK kinase activity, e.g. by contacting the cell with a serine/threoninekinase inhibitor. The cell may reside in culture or in situ, i.e. withinthe natural host. Preferred inhibitors are orally active in mammalianhosts. For diagnostic uses, the inhibitors or other IKK binding agentsare frequently labeled, such as with fluorescent, radioactive,chemiluminescent, or other easily detectable molecules, eitherconjugated directly to the binding agent or conjugated to a probespecific for the binding agent.

[0041] The amino acid sequences of the disclosed IKK-α polypeptides areused to back-translate IKK-α polypeptide-encoding nucleic acidsoptimized for selected expression systems (Holler et al. (1993) Gene136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used togenerate degenerate oligonucleotide primers and probes for use in theisolation of natural IKK-α-encoding nucleic acid sequences (“GCG”software, Genetics Computer Group, Inc, Madison Wis.). IKK-α-encodingnucleic acids used in IKK-α-expression vectors and incorporated intorecombinant host cells, e.g. for expression and screening, transgenicanimals, e.g. for functional studies such as the efficacy of candidatedrugs for disease associated with IKK-α-modulated cell function, etc.

[0042] The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a IKK-α cDNA specific sequencecomprising at least 12, preferably at least 24, more preferably at least36 and most preferably at least contiguous 96 bases of a strand of SEQID NO:3, particularly of SEQ ID NO:2, nucleotides 1-1913, and preferablyincluding at least one of bases 1-92, 1811, 1812, 1992, 1995, 2034,2035, 2039, 2040, 2050, 2055 and 2060, and sufficient to specificallyhybridize with a second nucleic acid comprising the complementary strandof SEQ ID NO:3 in the presence of a third nucleic acid comprising (SEQID NO:5). Demonstrating specific hybridization generally requiresstringent conditions, for example, hybridizing in a buffer comprising30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH 7.7, 0.001 MEDTA) buffer at a temperature of 42° C. and remaining bound when subjectto washing at 42° C. with 0.2×SSPE; preferably hybridizing in a buffercomprising 50% forynamide in 5×SSPE buffer at a temperature of 42° C.and remaining bound when subject to washing at 42° C. with 0.2×SSPEbuffer at 42° C. IKK-α nucleic acids can also be distinguished usingalignment algorithms, such as BLASTX (Altschul et al. (1990) Basic LocalAlignment Search Tool, J Mol Biol 215, 403-410).

[0043] In a particular embodiment, the invention provides IKK-αA¹⁶²⁸polynucleotides, comprising at least 18, 24, 36, 48, 72, 148, 356 or 728consecutive nucleotides of the nucleotide sequence set forth as SEQ IDNO:3, which consecutive polynucleotides comprise the polynucleotide 1628(A) of SEQ ID NO:3. Exemplary IKK-αA¹⁶²⁸ polynucleotides and allelespecific oligonucleotide probes having IKK-αGlu⁵⁴³ binding specificityand distinguishable by hybridization assays from IKK-αG¹⁶²⁸ are shown inTable IV. TABLE IV Exemplary IKK-αA¹⁶²⁸ polynucleotides havingIKK-αA¹⁶²⁸ binding specificity αΔ1 (SEQ ID NO: 3, nucleotides 1611-1628)αΔ2 (SEQ ID NO: 3, nucleotides 1628-1644) αΔ3 (SEQ ID NO: 3, nucleotides1620-1648) αΔ4 (SEQ ID NO: 3, nucleotides 1612-1648) αΔ5 (SEQ ID NO: 3,nucleotides 1608-1668) αΔ6 (SEQ ID NO: 3, nucleotides 1580-1650) αΔ7(SEQ ID NO: 3, nucleotides 1596-1636) αΔ8 (SEQ ID NO: 3, nucleotides1602-1687) αΔ9 (SEQ ID NO: 3, nucleotides 1614-1639) αΔ10 (SEQ ID NO: 3,nucleotides 1621-1753) αΔ11 (SEQ ID NO: 3, nucleotides 1502-1899) αΔ12(SEQ ID NO: 3, nucleotides 1020-1790)

[0044] The subject nucleic acids are of synthetic/non-natural sequencesand/or are isolated, i.e. unaccompanied by at least some of the materialwith which it is associated in its natural state, preferablyconstituting at least about 0.5%, preferably at least about 5% by weightof total nucleic acid present in a given fraction, and usuallyrecombinant, meaning they comprise a non-natural sequence or a naturalsequence joined to nucleotide(s) other than that which it is joined toon a natural chromosome. Recombinant nucleic acids comprising thenucleotide sequence of SEQ ID NO:3, or requisite fragments thereof,contain such sequence or fragment at a terminus, immediately flanked by(i.e. contiguous with) a sequence other than that which it is joined toon a natural chromosome, or flanked by a native flanking region fewerthan 10 kb, preferably fewer than 2 kb, which is at a terminus or isimmediately flanked by a sequence other than that which it is joined toon a natural chromosome. While the nucleic acids are usually RNA or DNA,it is often advantageous to use nucleic acids comprising other bases ornucleotide analogs to provide modified stability, etc.

[0045] The subject nucleic acids find a wide variety of applicationsincluding use as translatable transcripts, hybridization probes, PCRprimers, diagnostic nucleic acids, etc.; use in detecting the presenceof IKK-α genes and gene transcripts and in detecting or amplifyingnucleic acids encoding additional IKK-α homologs and structural analogs.In diagnosis, IKK-α hybridization probes find use in identifyingwild-type and mutant IKK-α alleles in clinical and laboratory samples.Mutant alleles are used to generate allele-specific oligonucleotide(ASO) probes for high-throughput clinical diagnoses. In therapy,therapeutic IKK-α nucleic acids are used to modulate cellular expressionor intracellular concentration or availability of active IKK-α.

[0046] The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of a IKKmodulatable cellular function. Generally, these screening methodsinvolve assaying for compounds which modulate IKK interaction with anatural IKK binding target, in particular, IKK phosphorylation ofIκB-derived substrates, particularly IκB and NIK substrates. A widevariety of assays for binding agents are provided including labeled invitro protein-protein binding assays, immunoassays, cell based assays,etc. The methods are amenable to automated, cost-effective highthroughput screening of chemical libraries for lead compounds.Identified reagents find use in the pharmaceutical industries for animaland human trials; for example, the reagents may be derivatized andrescreened in in vitro and in vivo assays to optimize activity andminimize toxicity for pharmaceutical development.

[0047] In vitro binding assays employ a mixture of components includingan IKK polypeptide, which may be part of a fusion product with anotherpeptide or polypeptide, e.g. a tag for detection or anchoring, etc. Theassay mixtures comprise a natural intracellular IKK binding target. In aparticular embodiment, the binding target is a substrate comprising IκBserines 32 and/or 36. Such substrates comprise a IκBα, β or ε peptideincluding the serine 32 and/or 36 residue and at least 5, preferably atleast 10, and more preferably at least 20 naturally occurringimmediately flanking residues on each side (e.g. for serine 36 peptides,residues 26-46, 22-42, or 12-32 or 151-171 for IκBα, β or ε-derivedsubstrates, respectively). While native full-length binding targets maybe used, it is frequently preferred to use portions (e.g. peptides)thereof so long as the portion provides binding affinity and avidity tothe subject IKK polypeptide conveniently measurable in the assay. Theassay mixture also comprises a candidate pharmacological agent.Candidate agents encompass numerous chemical classes, though typicallythey are organic compounds; preferably small organic compounds and areobtained from a wide variety of sources including libraries of syntheticor natural compounds. A variety of other reagents may also be includedin the mixture. These include reagents like ATP or ATP analogs (forkinase assays), salts, buffers, neutral proteins, e.g. albumin,detergents, protease inhibitors, nuclease inhibitors, antimicrobialagents, etc. may be used.

[0048] The resultant mixture is incubated under conditions whereby, butfor the presence of the candidate pharmacological agent, the IKKpolypeptide specifically binds the cellular binding target, portion oranalog with a reference binding affinity. The mixture components can beadded in any order that provides for the requisite bindings andincubations may be performed at any temperature which facilitatesoptimal binding. Incubation periods are likewise selected for optimalbinding but also minimized to facilitate rapid, high-throughputscreening.

[0049] After incubation, the agent-biased binding between the IKKpolypeptide and one or more binding targets is detected by anyconvenient way. For IKK kinase assays, ‘binding’ is generally detectedby a change in the phosphorylation of a IKK-α substrate. In thisembodiment, kinase activity may quantified by the transfer to thesubstrate of a labeled phosphate, where the label may provide for directdetection as radioactivity, luminescence, optical or electron density,etc. or indirect detection such as an epitope tag, etc. A variety ofmethods may be used to detect the label depending on the nature of thelabel and other assay components, e.g. through optical or electrondensity, radiative emissions, nonradiative energy transfers, etc. orindirectly detected with antibody conjugates, etc.

[0050] A difference in the binding affinity of the IKK polypeptide tothe target in the absence of the agent as compared with the bindingaffinity in the presence of the agent indicates that the agent modulatesthe binding of the IKK polypeptide to the IKK binding target.Analogously, in the cell-based assay also described below, a differencein IKK-α-dependent transcriptional activation in the presence andabsence of an agent indicates the agent modulates IKK function. Adifference, as used herein, is statistically significant and preferablyrepresents at least a 50%, more preferably at least a 90% difference.

[0051] The following experimental section and examples are offered byway of illustration and not by way of limitation.

Experimental

[0052] Identification of IKK-α

[0053] To investigate the mechanism of NIK-mediated NF-κB activation, weidentified proteins that associate directly with NIK by yeast two-hybridprotein interaction cloning (Fields and Song, 1989). An expressionvector was generated that encodes NIK fused to the DNA-binding domain ofthe yeast transcription factor GAL4. This vector was used as bait in atwo-hybrid screen of a human B cell cDNA library. From approximately sixmillion transformants, eight positive clones were obtained, asdetermined by activation of his and lacZ reporter genes. Of theseclones, three encoded a member of the TRAF family, TRAF3 (Hu et al.,1994; Cheng et al., 1995; Mosialos et al., 1995; Sato et al., 1995) andone encoded a novel protein we call IKK-α. Retransformation into yeastcells verified the interaction between NIK and IKK-α. A full-lengthhuman IKK-α clone was isolated by screening a Jurkat cDNA library with aprobe generated from the 5′-end of the IKK-α two-hybrid clone. IKK-αcomprises an N-terminal serine-threonine kinase catalytic domain, aC-terminal helix-loop-helix domain and a leucine zipper-like amphipathicα-helix juxtaposed in between the helix-loop-helix and kinase domain.

[0054] Interaction of IKK-α and NIK in Human Cells

[0055] The interaction of IKK-α with NIK was confirmed in mammalian cellcoimmunoprecipitation assays. Human IKK-α containing an N-terminal Flagepitope tag was transiently coexpressed in 293 human embryonic kidneycells with Myc epitope-tagged NIK or HA epitope-tagged TRAF proteins.Cell lysates were immunoprecipitated using a monoclonal antibody againstthe Flag epitope, and coprecipitating NIK or TRAF proteins were detectedby immunoblot analysis with an anti-Myc or anti-HA monoclonalantibodies. In this assay, IKK-α was able to coprecipitate NIKconfirming the interaction between both proteins as detected for IKK-αby yeast two-hybrid analysis. Also, a deletion mutant IKK-α proteinlacking most of the N-terminal kinase domain (IKK-α₍₃₀₇₋₇₄₅₎) was ableto associate with NIK, indicating that the α-helical C-terminal half ofIKK-α mediates the interaction with NIK. In contrast to NIK, IKK-αfailed to associate with either TRAF2 or TRAF3. However, when NIK wascoexpressed with IKK-α and TRAF2, strong coprecipitation of TRAF2 withIKK-α. was detected, indicating the formation of a ternary complexbetween IKK-α, NIK and TRAF2.

[0056] Effect of IKK-α and IKK-α Mutants on NF-κB Activation

[0057] To investigate a possible role for IKK-α in NF-κB activation, weexamined if transient overexpression of IKK-α might activate anNF-κB-dependent reporter gene. An E-selectin-luciferase reporterconstruct (Schindler and Baichwal, 1994) and a IKK-α expression vectorwere cotransfected into HeLa cells. IKK-α expression activated thereporter gene in a dose-dependent manner, with a maximal induction ofluciferase activity of about 6 to 7-fold compared to vector control.Similar results were obtained in 293 cells, where IKK-α overexpressioninduced reporter gene activity approximately 4-fold. TNF treatment didnot stimulate the weak NF-κB-inducing activity of overexpressed IKK-α inreporter gene assays. Thus, IKK-α induces NF-κB activation whenoverexpressed.

[0058] We next determined the effect of overexpression ofkinase-inactive IKK-α₍₃₀₇₋₇₄₅₎ that still associates with NIK onsignal-induced NF-κB activation in reporter gene assays in 293 cells.Overexpression of IKK-α₍₃₀₇₋₇₄₅₎ blocked TNF- and IL-1-induced reportergene activation similar to overexpression of NIK₍₆₂₄₋₉₄₇₎.IKK-α₍₃₀₇₋₇₄₅₎ was also found to inhibited NF-κB-dependent reporter geneactivity elicited by overexpression of TRAF2, TRAF6 and NIK. Takentogether these results demonstrate that a catalytically inactive IKK-αmutant is a dominant-negative inhibitor of TNF-, IL-1, TRAF- andNIK-induced NF-κB activation. This indicates that IKK-α functions as acommon mediator of NF-κB activation by TNF and IL-1 downstream of NIK.

PARENTHETICAL REFERENCES

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[0060] Baeuerle, P. A., and Henkel, T. (1994). Annu. Rev. Immunol. 12,141-179.

[0061] Beg, A. A., et al. (1993). Mol. Cell. Biol. 13, 3301-3310.

[0062] Cao, Z., Henzel, W. J., and Gao, X. (1996a). Science 271,1128-1131.

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[0064] Chen, Z., et al. (1995). Genes Dev. 9, 1586-1597.

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[0066] Connelly, M. A., and Marcu, K. B. (1995). Cell. Mol. Biol. Res.41, 537-549.

[0067] Dinarello, C. A. (1996). Biologic basis for interleukin-I indisease. Blood 87, 2095-2147.

[0068] Fields, S., and Song, O. -k. (1989). Nature 340, 245-246.

[0069] Finco, T. S., and Baldwin, A. S. (1995). Immunity 3, 263-272.

[0070] Gedrich, R. W., et al. (1996). J. Biol. Chem. 271, 12852-12858.

[0071] Hill, C. S., and Treisman, R. (1995). Cell 80, 199-211.

[0072] Hsu, H., Shu, H. -B., Pan, M. -P., and Goeddel, D. V. (1996).Cell 84, 299-308.

[0073] Hu, H. M., et al. (1994). J. Biol. Chem. 269, 30069-30072.

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[0076] Malinin, N. L., et al. (1997). Nature 385, 540-544.

[0077] Mock et al. (1995). Genomics 27, 348-351.

[0078] Mosialos, G., et al. (1995). Cell 80, 389-399.

[0079] Nakano, H., et al. (1996). J. Biol. Chem. 271, 14661-14664.

[0080] Osbom, L., Kunkel, S., and Nabel, G. J. (1989). Proc. Natl. Acad.Sci. U.S. Pat. No. 86,2336-2340.

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[0088] Verma, I. M., et al. (1995). Genes Dev. 9, 2723-2735.

EXAMPLES

[0089] 1. Protocol for at IKK-α-IκBα phosphorylation assay.

[0090] A. Reagents:

[0091] Neutralite Avidin: 20 μg/ml in PBS.

[0092] kinase: ₁₀ ⁻⁸-10⁻⁵ M IKK-α (SEQ ID NO:4) at 20 μg/ml in PBS.

[0093] substrate: 10⁻⁷-10⁻⁴ M biotinylated substrate (21 residue peptideconsisting of residues 26-46 of human IκBα) at 40 μg/ml in PBS.

[0094] Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

[0095] Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH7.4, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA,cocktail of protease inhibitors.

[0096] [³²P]γ-ATP 10× stock: 2×10⁻⁵ M cold ATP with 100 μCi [³²P]γ-ATP.Place in the 4° C. microfridge during screening.

[0097] Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2 mM NaVo₃(Sigma #S-6508) in 10 ml of PBS.

[0098] B. Preparation of Assay Plates:

[0099] Coat with 120 μl of stock N Avidin per well overnight at 4° C.

[0100] Wash 2 times with 200 μl PBS.

[0101] Block with 150 μL of blocking buffer.

[0102] Wash 2 times with 200 μl PBS.

[0103] C. Assay:

[0104] Add 40 μl assay buffer/well.

[0105] Add 40 μl biotinylated substrate (2-200 pmoles/40 ul in assaybuffer)

[0106] Add 40 μl kinase (0.1-10 pmoles/40 ul in assay buffer)

[0107] Add 10 μl compound or extract.

[0108] Add 10 μl [³²P]γ-ATP 10× stock.

[0109] Shake at 25° C. for 15 minutes.

[0110] Incubate additional 45 minutes at 25° C.

[0111] Stop the reaction by washing 4 times with 200 μl PBS.

[0112] Add 150 μl scintillation cocktail.

[0113] Count in Topcount.

[0114] D. Controls for All Assays (Located on Each Plate):

[0115] a. Non-specific binding

[0116] b. cold ATP at 80% inhibition.

[0117] 2. Protocol for High Throughput IKK-α-NIK Binding Assay.

[0118] A. Reagents:

[0119] Neutralite Avidin: 20 μg/ml in PBS.

[0120] Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

[0121] Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1%glycerol, 0.5% NP-40, 50 mM b-mercaptoethanol, 1 mg/ml BSA, cocktail ofprotease inhibitors.

[0122]³³P IKK-α polypeptide 10× stock: 10⁻⁸-10⁻⁶ M “cold” IKK-αsupplemented with 200,000-250,000 cpm of labeled IKK-α (Beckmancounter). Place in the 4° C. microfridge during screening.

[0123] Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB#109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma#B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and2 mM NaVO₃(Sigma #S-6508) in 10 ml of PBS.

[0124] NIK: 10⁻⁷-10⁻⁵ M biotinylated NIK in PBS.

[0125] B. Preparation of Assay Plates:

[0126] Coat with 120 μl of stock N-Avidin per well overnight at 4 C.

[0127] Wash 2 times with 200 μl PBS.

[0128] Block with 150 μL of blocking buffer.

[0129] Wash 2 times with 200 μl PBS.

[0130] C. Assay:

[0131] Add 40 μl assay buffer/well.

[0132] Add 10 μl compound or extract.

[0133] Add 10 μl ³³P-IKK-α (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹-10⁻⁷ Mfinal conc).

[0134] Shake at 25° C. for 15 minutes.

[0135] Incubate additional 45 minutes at 25° C.

[0136] Add 40 μM biotinylated NIK (0.1-10 pmoles/40 ul in assay buffer)

[0137] Incubate 1 hour at room temperature.

[0138] Stop the reaction by washing 4 times with 200 μM PBS.

[0139] Add 150 μM scintillation cocktail.

[0140] Count in Topcount.

[0141] D. Controls for All Assays (Located on Each Plate):

[0142] a. Non-specific binding

[0143] b. Soluble (non-biotinylated NIK) at 80% inhibition.

[0144] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.                    #               SEQUENCE  #LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 5(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 2268 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #1: ATGAGCTGGT CACCTTCCCT GACAACGCAG ACATGTGGGG CCTGGGAAAT GA#AAGAGCGC     60CTTGGGACAG GGGGATTTGG AAATGTCATC CGATGGCACA ATCAGGAAAC AG#GTGAGCAG    120ATTGCCATCA AGCAGTGCCG GCAGGAGCTC AGCCCCCGGA ACCGAGAGCG GT#GGTGCCTG    180GAGATCCAGA TCATGAGAAG GCTGACCCAC CCCAATGTGG TGGCTGCCCG AG#ATGTCCCT    240GAGGGGATGC AGAACTTGGC GCCCAATGAC CTGCCCCTGC TGGCCATGGA GT#ACTGCCAA    300GGAGGAGATC TCCGGAAGTA CCTGAACCAG TTTGAGAACT GCTGTGGTCT GC#GGGAAGGT    360GCCATCCTCA CCTTGCTGAG TGACATTGCC TCTGCGCTTA GATACCTTCA TG#AAAACAGA    420ATCATCCATC GGGATCTAAA GCCAGAAAAC ATCGTCCTGC AGCAAGGAGA AC#AGAGGTTA    480ATACACAAAA TTATTGACCT AGGATATGCC AAGGAGCTGG ATCAGGGCAG TC#TTTGCACA    540TCATTCGTGG GGACCCTGCA GTACCTGGCC CCAGAGCTAC TGGAGCAGCA GA#AGTACACA    600GTGACCGTCG ACTACTGGAG CTTCGGCACC CTGGCCTTTG AGTGCATCAC GG#GCTTCCGG    660CCCTTCCTCC CCAACTGGCA GCCCGTGCAG TGGCATTCAA AAGTGCGGCA GA#AGAGTGAG    720GTGGACATTG TTGTTAGCGA AGACTTGAAT GGAACGGTGA AGTTTTCAAG CT#CTTTACCC    780TACCCCAATA ATCTTAACAG TGTCCTGGCT GAGCGACTGG AGAAGTGGCT GC#AACTGATG    840CTGATGTGGC ACCCCCGACA GAGGGGCACG GATCCCACGT ATGGGCCCAA TG#GCTGCTTC    900AAGGCCCTGG ATGACATCTT AAACTTAAAG CTGGTTCATA TCTTGAACAT GG#TCACGGGC    960ACCATCCACA CCTACCCTGT GACAGAGGAT GAGAGTCTGC AGAGCTTGAA GG#CCAGAATC   1020CAACAGGACA CGGGCATCCC AGAGGAGGAC CAGGAGCTGC TGCAGGAAGC GG#GCCTGGCG   1080TTGATCCCCG ATAAGCCTGC CACTCAGTGT ATTTCAGACG GCAAGTTAAA TG#AGGGCCAC   1140ACATTGGACA TGGATCTTGT TTTTCTCTTT GACAACAGTA AAATCACCTA TG#AGACTCAG   1200ATCTCCCCAC GGCCCCAACC TGAAAGTGTC AGCTGTATCC TTCAAGAGCC CA#AGAGGAAT   1260CTCGCCTTCT TCCAGCTGAG GAAGGTGTGG GGCCAGGTCT GGCACAGCAT CC#AGACCCTG   1320AAGGAAGATT GCAACCGGCT GCAGCAGGGA CAGCGAGCCG CCATGATGAA TC#TCCTCCGA   1380AACAACAGCT GCCTCTCCAA AATGAAGAAT TCCATGGCTT CCATGTCTCA GC#AGCTCAAG   1440GCCAAGTTGG ATTTCTTCAA AACCAGCATC CAGATTGACC TGGAGAAGTA CA#GCGAGCAA   1500ACCGAGTTTG GGATCACATC AGATAAACTG CTGCTGGCCT GGAGGGAAAT GG#AGCAGGCT   1560GTGGAGCTCT GTGGGCGGGA GAACGAAGTG AAACTCCTGG TAGAACGGAT GA#TGGCTCTG   1620CAGACCGACA TTGTGGACTT ACAGAGGAGC CCCATGGGCC GGAAGCAGGG GG#GAACGCTG   1680GACGACCTAG AGGAGCAAGC AAGGGAGCTG TACAGGAGAC TAAGGGAAAA AC#CTCGAGAC   1740CAGCGAACTG AGGGTGACAG TCAGGAAATG GTACGGCTGC TGCTTCAGGC AA#TTCAGAGC   1800TTCGAGAAGA AAGTGCGAGT GATCTATACG CAGCTCAGTA AAACTGTGGT TT#GCAAGCAG   1860AAGGCGCTGG AACTGTTGCC CAAGGTGGAA GAGGTGGTGA GCTTAATGAA TG#AGGATGAG   1920AAGACTGTTG TCCGGCTGCA GGAGAAGCGG CAGAAGGAGC TCTGGAATCT CC#TGAAGATT   1980GCTTGTAGCA AGGTCCGTGG TCCTGTCAGT GGAAGCCCGG ATAGCATGAA TG#CCTCTCGA   2040CTTAGCCAGC CTGGGCAGCT GATGTCTCAG CCCTCCACGG CCTCCAACAG CT#TACCTGAG   2100CCAGCCAAGA AGAGTGAAGA ACTGGTGGCT GAAGCACATA ACCTCTGCAC CC#TGCTAGAA   2160AATGCCATAC AGGACACTGT GAGGGAACAA GACCAGAGTT TCACGGCCCT AG#ACTGGAGC   2220 TGGTTACAGA CGGAAGAAGA AGAGCACAGC TGCCTGGAGC AGGCCTCA  #              2268 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 756 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Met Ser Trp Ser Pro Ser Leu Thr Thr Gln Th #r Cys Gly Ala Trp Glu1               5    #                10   #                15Met Lys Glu Arg Leu Gly Thr Gly Gly Phe Gl #y Asn Val Ile Arg Trp            20       #            25       #            30His Asn Gln Glu Thr Gly Glu Gln Ile Ala Il #e Lys Gln Cys Arg Gln        35           #        40           #        45Glu Leu Ser Pro Arg Asn Arg Glu Arg Trp Cy #s Leu Glu Ile Gln Ile    50               #    55               #    60Met Arg Arg Leu Thr His Pro Asn Val Val Al #a Ala Arg Asp Val Pro65                   #70                   #75                   #80Glu Gly Met Gln Asn Leu Ala Pro Asn Asp Le #u Pro Leu Leu Ala Met                85   #                90   #                95Glu Tyr Cys Gln Gly Gly Asp Leu Arg Lys Ty #r Leu Asn Gln Phe Glu            100       #           105       #           110Asn Cys Cys Gly Leu Arg Glu Gly Ala Ile Le #u Thr Leu Leu Ser Asp        115           #       120           #       125Ile Ala Ser Ala Leu Arg Tyr Leu His Glu As #n Arg Ile Ile His Arg    130               #   135               #   140Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gl #n Gly Glu Gln Arg Leu145                 1 #50                 1 #55                 1 #60Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Ly #s Glu Leu Asp Gln Gly                165   #               170   #               175Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gl #n Tyr Leu Ala Pro Glu            180       #           185       #           190Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Va #l Asp Tyr Trp Ser Phe        195           #       200           #       205Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Ph #e Arg Pro Phe Leu Pro    210               #   215               #   220Asn Trp Gln Pro Val Gln Trp His Ser Lys Va #l Arg Gln Lys Ser Glu225                 2 #30                 2 #35                 2 #40Val Asp Ile Val Val Ser Glu Asp Leu Asn Gl #y Thr Val Lys Phe Ser                245   #               250   #               255Ser Ser Leu Pro Tyr Pro Asn Asn Leu Asn Se #r Val Leu Ala Glu Arg            260       #           265       #           270Leu Glu Lys Trp Leu Gln Leu Met Leu Met Tr #p His Pro Arg Gln Arg        275           #       280           #       285Gly Thr Asp Pro Thr Tyr Gly Pro Asn Gly Cy #s Phe Lys Ala Leu Asp    290               #   295               #   300Asp Ile Leu Asn Leu Lys Leu Val His Ile Le #u Asn Met Val Thr Gly305                 3 #10                 3 #15                 3 #20Thr Ile His Thr Tyr Pro Val Thr Glu Asp Gl #u Ser Leu Gln Ser Leu                325   #               330   #               335Lys Ala Arg Ile Gln Gln Asp Thr Gly Ile Pr #o Glu Glu Asp Gln Glu            340       #           345       #           350Leu Leu Gln Glu Ala Gly Leu Ala Leu Ile Pr #o Asp Lys Pro Ala Thr        355           #       360           #       365Gln Cys Ile Ser Asp Gly Lys Leu Asn Glu Gl #y His Thr Leu Asp Met    370               #   375               #   380Asp Leu Val Phe Leu Phe Asp Asn Ser Lys Il #e Thr Tyr Glu Thr Gln385                 3 #90                 3 #95                 4 #00Ile Ser Pro Arg Pro Gln Pro Glu Ser Val Se #r Cys Ile Leu Gln Glu                405   #               410   #               415Pro Lys Arg Asn Leu Ala Phe Phe Gln Leu Ar #g Lys Val Trp Gly Gln            420       #           425       #           430Val Trp His Ser Ile Gln Thr Leu Lys Glu As #p Cys Asn Arg Leu Gln        435           #       440           #       445Gln Gly Gln Arg Ala Ala Met Met Asn Leu Le #u Arg Asn Asn Ser Cys    450               #   455               #   460Leu Ser Lys Met Lys Asn Ser Met Ala Ser Me #t Ser Gln Gln Leu Lys465                 4 #70                 4 #75                 4 #80Ala Lys Leu Asp Phe Phe Lys Thr Ser Ile Gl #n Ile Asp Leu Glu Lys                485   #               490   #               495Tyr Ser Glu Gln Thr Glu Phe Gly Ile Thr Se #r Asp Lys Leu Leu Leu            500       #           505       #           510Ala Trp Arg Glu Met Glu Gln Ala Val Glu Le #u Cys Gly Arg Glu Asn        515           #       520           #       525Glu Val Lys Leu Leu Val Glu Arg Met Met Al #a Leu Gln Thr Asp Ile    530               #   535               #   540Val Asp Leu Gln Arg Ser Pro Met Gly Arg Ly #s Gln Gly Gly Thr Leu545                 5 #50                 5 #55                 5 #60Asp Asp Leu Glu Glu Gln Ala Arg Glu Leu Ty #r Arg Arg Leu Arg Glu                565   #               570   #               575Lys Pro Arg Asp Gln Arg Thr Glu Gly Asp Se #r Gln Glu Met Val Arg            580       #           585       #           590Leu Leu Leu Gln Ala Ile Gln Ser Phe Glu Ly #s Lys Val Arg Val Ile        595           #       600           #       605Tyr Thr Gln Leu Ser Lys Thr Val Val Cys Ly #s Gln Lys Ala Leu Glu    610               #   615               #   620Leu Leu Pro Lys Val Glu Glu Val Val Ser Le #u Met Asn Glu Asp Glu625                 6 #30                 6 #35                 6 #40Lys Thr Val Val Arg Leu Gln Glu Lys Arg Gl #n Lys Glu Leu Trp Asn                645   #               650   #               655Leu Leu Lys Ile Ala Cys Ser Lys Val Arg Gl #y Pro Val Ser Gly Ser            660       #           665       #           670Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gl #n Pro Gly Gln Leu Met        675           #       680           #       685Ser Gln Pro Ser Thr Ala Ser Asn Ser Leu Pr #o Glu Pro Ala Lys Lys    690               #   695               #   700Ser Glu Glu Leu Val Ala Glu Ala His Asn Le #u Cys Thr Leu Leu Glu705                 7 #10                 7 #15                 7 #20Asn Ala Ile Gln Asp Thr Val Arg Glu Gln As #p Gln Ser Phe Thr Ala                725   #               730   #               735Leu Asp Trp Ser Trp Leu Gln Thr Glu Glu Gl #u Glu His Ser Cys Leu            740       #           745       #           750Glu Gln Ala Ser         755 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 2238 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #3: ATGGAGCGGC CCCCGGGGCT GCGGCCGGGC GCGGGCGGGC CCTGGGAGAT GC#GGGAGCGG     60CTGGGCACCG GCGGCTTCGG GAACGTCTGT CTGTACCAGC ATCGGGAACT TG#ATCTCAAA    120ATAGCAATTA AGTCTTGTCG CCTAGAGCTA AGTACCAAAA ACAGAGAACG AT#GGTGCCAT    180GAAATCCAGA TTATGAAGAA GTTGAACCAT GCCAATGTTG TAAAGGCCTG TG#ATGTTCCT    240GAAGAATTGA ATATTTTGAT TCATGATGTG CCTCTTCTAG CAATGGAATA CT#GTTCTGGA    300GGAGATCTCC GAAAGCTGCT CAACAAACCA GAAAATTGTT GTGGACTTAA AG#AAAGCCAG    360ATACTTTCTT TACTAAGTGA TATAGGGTCT GGGATTCGAT ATTTGCATGA AA#ACAAAATT    420ATACATCGAG ATCTAAAACC TGAAAACATA GTTCTTCAGG ATGTTGGTGG AA#AGATAATA    480CATAAAATAA TTGATCTGGG ATATGCCAAA GATGTTGATC AAGGAAGTCT GT#GTACATCT    540TTTGTGGGAA CACTGCAGTA TCTGGCCCCA GAGCTCTTTG AGAATAAGCC TT#ACACAGCC    600ACTGTTGATT ATTGGAGCTT TGGGACCATG GTATTTGAAT GTATTGCTGG AT#ATAGGCCT    660TTTTTGCATC ATCTGCAGCC ATTTACCTGG CATGAGAAGA TTAAGAAGAA GG#ATCCAAAG    720TGTATATTTG CATGTGAAGA GATGTCAGGA GAAGTTCGGT TTAGTAGCCA TT#TACCTCAA    780CCAAATAGCC TTTGTAGTTT AATAGTAGAA CCCATGGAAA ACTGGCTACA GT#TGATGTTG    840AATTGGGACC CTCAGCAGAG AGGAGGACCT GTTGACCTTA CTTTGAAGCA GC#CAAGATGT    900TTTGTATTAA TGGATCACAT TTTGAATTTG AAGATAGTAC ACATCCTAAA TA#TGACTTCT    960GCAAAGATAA TTTCTTTTCT GTTACCACCT GATGAAAGTC TTCATTCACT AC#AGTCTCGT   1020ATTGAGCGTG AAACTGGAAT AAATACTGGT TCTCAAGAAC TTCTTTCAGA GA#CAGGAATT   1080TCTCTGGATC CTCGGAAACC AGCCTCTCAA TGTGTTCTAG ATGGAGTTAG AG#GCTGTGAT   1140AGCTATATGG TTTATTTGTT TGATAAAAGT AAAACTGTAT ATGAAGGGCC AT#TTGCTTCC   1200AGAAGTTTAT CTGATTGTGT AAATTATATT GTACAGGACA GCAAAATACA GC#TTCCAATT   1260ATACAGCTGC GTAAAGTGTG GGCTGAAGCA GTGCACTATG TGTCTGGACT AA#AAGAAGAC   1320TATAGCAGGC TCTTTCAGGG ACAAAGGGCA GCAATGTTAA GTCTTCTTAG AT#ATAATGCT   1380AACTTAACAA AAATGAAGAA CACTTTGATC TCAGCATCAC AACAACTGAA AG#CTAAATTG   1440GAGTTTTTTC ACAAAAGCAT TCAGCTTGAC TTGGAGAGAT ACAGCGAGCA GA#TGACGTAT   1500GGGATATCTT CAGAAAAAAT GCTAAAAGCA TGGAAAGAAA TGGAAGAAAA GG#CCATCCAC   1560TATGCTGAGG TTGGTGTCAT TGGATACCTG GAGGATCAGA TTATGTCTTT GC#ATGCTGAA   1620ATCATGGAGC TACAGAAGAG CCCCTATGGA AGACGTCAGG GAGACTTGAT GG#AATCTCTG   1680GAACAGCGTG CCATTGATCT ATATAAGCAG TTAAAACACA GACCTTCAGA TC#ACTCCTAC   1740AGTGACAGCA CAGAGATGGT GAAAATCATT GTGCACACTG TGCAGAGTCA GG#ACCGTGTG   1800CTCAAGGAGC TGTTTGGTCA TTTGAGCAAG TTGTTGGGCT GTAAGCAGAA GA#TTATTGAT   1860CTACTCCCTA AGGTGGAAGT GGCCCTCAGT AATATCAAAG AAGCTGACAA TA#CTGTCATG   1920TTCATGCAGG GAAAAAGGCA GAAAGAAATA TGGCATCTCC TTAAAATTGC CT#GTACACAG   1980AGTTCTGCCC GGTCCCTTGT AGGATCCAGT CTAGAAGGTG CAGTAACCCC TC#AGACATCA   2040GCATGGCTGC CCCCGACTTC AGCAGAACAT GATCATTCTC TGTCATGTGT GG#TAACTCCT   2100CAAGATGGGG AGACTTCAGC ACAAATGATA GAAGAAAATT TGAACTGCCT TG#GCCATTTA   2160AGCACTATTA TTCATGAGGC AAATGAGGAA CAGGGCAATA GTATGATGAA TC#TTGATTGG   2220 AGTTGGTTAA CAGAATGA              #                  #                   #2238 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 745 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:Met Glu Arg Pro Pro Gly Leu Arg Pro Gly Al #a Gly Gly Pro Trp Glu 1               5   #                10   #                15Met Arg Glu Arg Leu Gly Thr Gly Gly Phe Gl #y Asn Val Cys Leu Tyr             20      #             25      #             30Gln His Arg Glu Leu Asp Leu Lys Ile Ala Il #e Lys Ser Cys Arg Leu         35          #         40          #         45Glu Leu Ser Thr Lys Asn Arg Glu Arg Trp Cy #s His Glu Ile Gln Ile     50              #     55              #     60Met Lys Lys Leu Asn His Ala Asn Val Val Ly #s Ala Cys Asp Val Pro 65                  # 70                  # 75                  # 80Glu Glu Leu Asn Ile Leu Ile His Asp Val Pr #o Leu Leu Ala Met Glu                 85  #                 90  #                 95Tyr Cys Ser Gly Gly Asp Leu Arg Lys Leu Le #u Asn Lys Pro Glu Asn            100       #           105       #           110Cys Cys Gly Leu Lys Glu Ser Gln Ile Leu Se #r Leu Leu Ser Asp Ile        115           #       120           #       125Gly Ser Gly Ile Arg Tyr Leu His Glu Asn Ly #s Ile Ile His Arg Asp    130               #   135               #   140Leu Lys Pro Glu Asn Ile Val Leu Gln Asp Va #l Gly Gly Lys Ile Ile145                 1 #50                 1 #55                 1 #60His Lys Ile Ile Asp Leu Gly Tyr Ala Lys As #p Val Asp Gln Gly Ser                165   #               170   #               175Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Ty #r Leu Ala Pro Glu Leu            180       #           185       #           190Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val As #p Tyr Trp Ser Phe Gly        195           #       200           #       205Thr Met Val Phe Glu Cys Ile Ala Gly Tyr Ar #g Pro Phe Leu His His    210               #   215               #   220Leu Gln Pro Phe Thr Trp His Glu Lys Ile Ly #s Lys Lys Asp Pro Lys225                 2 #30                 2 #35                 2 #40Cys Ile Phe Ala Cys Glu Glu Met Ser Gly Gl #u Val Arg Phe Ser Ser                245   #               250   #               255His Leu Pro Gln Pro Asn Ser Leu Cys Ser Le #u Ile Val Glu Pro Met            260       #           265       #           270Glu Asn Trp Leu Gln Leu Met Leu Asn Trp As #p Pro Gln Gln Arg Gly        275           #       280           #       285Gly Pro Val Asp Leu Thr Leu Lys Gln Pro Ar #g Cys Phe Val Leu Met    290               #   295               #   300Asp His Ile Leu Asn Leu Lys Ile Val His Il #e Leu Asn Met Thr Ser305                 3 #10                 3 #15                 3 #20Ala Lys Ile Ile Ser Phe Leu Leu Pro Pro As #p Glu Ser Leu His Ser                325   #               330   #               335Leu Gln Ser Arg Ile Glu Arg Glu Thr Gly Il #e Asn Thr Gly Ser Gln            340       #           345       #           350Glu Leu Leu Ser Glu Thr Gly Ile Ser Leu As #p Pro Arg Lys Pro Ala        355           #       360           #       365Ser Gln Cys Val Leu Asp Gly Val Arg Gly Cy #s Asp Ser Tyr Met Val    370               #   375               #   380Tyr Leu Phe Asp Lys Ser Lys Thr Val Tyr Gl #u Gly Pro Phe Ala Ser385                 3 #90                 3 #95                 4 #00Arg Ser Leu Ser Asp Cys Val Asn Tyr Ile Va #l Gln Asp Ser Lys Ile                405   #               410   #               415Gln Leu Pro Ile Ile Gln Leu Arg Lys Val Tr #p Ala Glu Ala Val His            420       #           425       #           430Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Ar #g Leu Phe Gln Gly Gln        435           #       440           #       445Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr As #n Ala Asn Leu Thr Lys    450               #   455               #   460Met Lys Asn Thr Leu Ile Ser Ala Ser Gln Gl #n Leu Lys Ala Lys Leu465                 4 #70                 4 #75                 4 #80Glu Phe Phe His Lys Ser Ile Gln Leu Asp Le #u Glu Arg Tyr Ser Glu                485   #               490   #               495Gln Met Thr Tyr Gly Ile Ser Ser Glu Lys Me #t Leu Lys Ala Trp Lys            500       #           505       #           510Glu Met Glu Glu Lys Ala Ile His Tyr Ala Gl #u Val Gly Val Ile Gly        515           #       520           #       525Tyr Leu Glu Asp Gln Ile Met Ser Leu His Al #a Glu Ile Met Glu Leu    530               #   535               #   540Gln Lys Ser Pro Tyr Gly Arg Arg Gln Gly As #p Leu Met Glu Ser Leu545                 5 #50                 5 #55                 5 #60Glu Gln Arg Ala Ile Asp Leu Tyr Lys Gln Le #u Lys His Arg Pro Ser                565   #               570   #               575Asp His Ser Tyr Ser Asp Ser Thr Glu Met Va #l Lys Ile Ile Val His            580       #           585       #           590Thr Val Gln Ser Gln Asp Arg Val Leu Lys Gl #u Leu Phe Gly His Leu        595           #       600           #       605Ser Lys Leu Leu Gly Cys Lys Gln Lys Ile Il #e Asp Leu Leu Pro Lys    610               #   615               #   620Val Glu Val Ala Leu Ser Asn Ile Lys Glu Al #a Asp Asn Thr Val Met625                 6 #30                 6 #35                 6 #40Phe Met Gln Gly Lys Arg Gln Lys Glu Ile Tr #p His Leu Leu Lys Ile                645   #               650   #               655Ala Cys Thr Gln Ser Ser Ala Arg Ser Leu Va #l Gly Ser Ser Leu Glu            660       #           665       #           670Gly Ala Val Thr Pro Gln Thr Ser Ala Trp Le #u Pro Pro Thr Ser Ala        675           #       680           #       685Glu His Asp His Ser Leu Ser Cys Val Val Th #r Pro Gln Asp Gly Glu    690               #   695               #   700Thr Ser Ala Gln Met Ile Glu Glu Asn Leu As #n Cys Leu Gly His Leu705                 7 #10                 7 #15                 7 #20Ser Thr Ile Ile His Glu Ala Asn Glu Glu Gl #n Gly Asn Ser Met Met                725   #               730   #               735Asn Leu Asp Trp Ser Trp Leu Thr Glu             740      #           745 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 2146 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #5: GTACCAGCAT CGGGAACTTG ATCTCAAAAT AGCAATTAAG TCTTGTCGCC TA#GAGCTAAG     60TACCAAAAAC AGAGAACGAT GGTGCCATGA AATCCAGATT ATGAAGAAGT TG#AACCATGC    120CAATGTTGTA AAGGCCTGTG ATGTTCCTGA AGAATTGAAT ATTTTGATTC AT#GATGTGCC    180TCTTCTAGCA ATGGAATACT GTTCTGGAGG AGATCTCCGA AAGCTGCTCA AC#AAACCAGA    240AAATTGTTGT GGACTTAAAG AAAGCCAGAT ACTTTCTTTA CTAAGTGATA TA#GGGTCTGG    300GATTCGATAT TTGCATGAAA ACAAAATTAT ACATCGAGAT CTAAAACCTG AA#AACATAGT    360TCTTCAGGAT GTTGGTGGAA AGATAATACA TAAAATAATT GATCTGGGAT AT#GCCAAAGA    420TGTTGATCAA GGAAGTCTGT GTACATCTTT TGTGGGAACA CTGCAGTATC TG#GCCCCAGA    480GCTCTTTGAG AATAAGCCTT ACACAGCCAC TGTTGATTAT TGGAGCTTTG GG#ACCATGGT    540ATTTGAATGT ATTGCTGGAT ATAGGCCTTT TTTGCATCAT CTGCAGCCAT TT#ACCTGGCA    600TGAGAAGATT AAGAAGAAGG ATCCAAAGTG TATATTTGCA TGTGAAGAGA TG#TCAGGAGA    660AGTTCGGTTT AGTAGCCATT TACCTCAACC AAATAGCCTT TGTAGTTTAA TA#GTAGAACC    720CATGGAAAAC TGGCTACAGT TGATGTTGAA TTGGGACCCT CAGCAGAGAG GA#GGACCTGT    780TGACCTTACT TTGAAGCAGC CAAGATGTTT TGTATTAATG GATCACATTT TG#AATTTGAA    840GATAGTACAC ATCCTAAATA TGACTTCTGC AAAGATAATT TCTTTTCTGT TA#CCACCTGA    900TGAAAGTCTT CATTCACTAC AGTCTCGTAT TGAGCGTGAA ACTGGAATAA AT#ACTGGTTC    960TCAAGAACTT CTTTCAGAGA CAGGAATTTC TCTGGATCCT CGGAAACCAG CC#TCTCAATG   1020TGTTCTAGAT GGAGTTAGAG GCTGTGATAG CTATATGGTT TATTTGTTTG AT#AAAAGTAA   1080AACTGTATAT GAAGGGCCAT TTGCTTCCAG AAGTTTATCT GATTGTGTAA AT#TATATTGT   1140ACAGGACAGC AAAATACAGC TTCCAATTAT ACAGCTGCGT AAAGTGTGGG CT#GAAGCAGT   1200GCACTATGTG TCTGGACTAA AAGAAGACTA TAGCAGGCTC TTTCAGGGAC AA#AGGGCAGC   1260AATGTTAAGT CTTCTTAGAT ATAATGCTAA CTTAACAAAA ATGAAGAACA CT#TTGATCTC   1320AGCATCACAA CAACTGAAAG CTAAATTGGA GTTTTTTCAC AAAAGCATTC AG#CTTGACTT   1380GGAGAGATAC AGCGAGCAGA TGACGTATGG GATATCTTCA GAAAAAATGC TA#AAAGCATG   1440GAAAGAAATG GAAGAAAAGG CCATCCACTA TGCTGAGGTT GGTGTCATTG GA#TACCTGGA   1500GGATCAGATT ATGTCTTTGC ATGCTGAAAT CATGGAGCTA CAGAAGAGCC CC#TATGGAAG   1560ACGTCAGGGA GACTTGATGG AATCTCTGGA ACAGCGTGCC ATTGATCTAT AT#AAGCAGTT   1620AAAACACAGA CCTTCAGATC ACTCCTACAG TGACAGCACA GAGATGGTGA AA#ATCATTGT   1680GCACACTGTG CAGAGTCAGG ACCGTGTGCT CAAGGAGCGT TTTGGTCATT TG#AGCAAGTT   1740GTTGGGCTGT AAGCAGAAGA TTATTGATCT ACTCCCTAAG GTGGAAGTGG CC#CTCAGTAA   1800TATCAAAGAA GCTGACAATA CTGTCATGTT CATGCAGGGA AAAAGGCAGA AA#GAAATATG   1860GCATCTCCTT AAAATTGCCT GTACACAGAG TTCTGCCCGC TCTCTTGTAG GA#TCCAGTCT   1920AGAAGGTGCA GTAACCCCTC AAGCATACGC ATGGCTGGCC CCCGACTTAG CA#GAACATGA   1980TCATTCTCTG TCATGTGTGG TAACTCCTCA AGATGGGGAG ACTTCAGCAC AA#ATGATAGA   2040AGAAAATTTG AACTGCCTTG GCCATTTAAG CACTATTATT CATGAGGCAA AT#GAGGAACA   2100 GGGCAATAGT ATGATGAATC TTGATTGGAG TTGGTTAACA GAATGA   #               2146

What is claimed is:
 1. An isolated IKK-αGlu⁵⁴³ polypeptide, comprisingat least 10 consecutive amino acid residues of the amino acid sequenceset forth as SEQ ID NO:4, which consecutive amino acid residues comprisethe amino acid residue 543 (Glu) of SEQ ID NO:4.
 2. An isolatedpolypeptide according to claim 1, wherein said polypeptide has anactivity selected from at least one of: a kinase or kinase inhibitoryactivity, a NIK-binding or binding inhibitory activity, an IκB-bindingor binding inhibitory activity and an NFκB activating or inhibitoryactivity.
 3. An isolated or recombinant IKK-αA¹⁶²⁸ nucleic acidcomprising at least 24 consecutive nucleotides of the nucleotidesequence set forth as SEQ ID NO:3, which consecutive polynucleotidescomprise the polynucleotide 1628 (A) of SEQ ID NO:3.
 4. A recombinantnucleic acid encoding a polypeptide according to claim
 1. 5. A cellcomprising a nucleic acid according to claim
 4. 6. A method of making anisolated polypeptide according to claim 1, said method comprising steps:introducing a nucleic acid according to claim 4 into a host cell orcellular extract, incubating said host cell or extract under conditionswhereby said nucleic acid is expressed as a transcript and saidtranscript is expressed as a translation product comprising saidpolypeptide, and isolating said translation product.
 7. A method ofscreening for an agent which modulates the interaction of an IKKpolypeptide to a binding target, said method comprising the steps of:incubating a mixture comprising: an isolated polypeptide according toclaim 1, a binding target of said polypeptide, and a candidate agent;under conditions whereby, but for the presence of said agent, saidpolypeptide specifically binds said binding target at a referenceaffinity; detecting the binding affinity of said polypeptide to saidbinding target to determine an agent-biased affinity, wherein adifference between the agent-biased affinity and the reference affinityindicates that said agent modulates the binding of said polypeptide tosaid binding target.
 8. A method according to claim 7, wherein saidbinding target is a natural intracellular substrate and said referenceand agent-biased binding affinity is detected as phosphorylation of saidsubstrate.
 9. A method of screening for an agent which modulates theinteraction of an IKK polypeptide to a binding target, said methodcomprising the steps of: incubating a mixture comprising: an isolatedpolypeptide comprising SEQ ID NO: 2 or 4, or a deletion mutant thereofretaining IκB kinase activity, an IκB polypeptide comprising at least asix residue domain of a natural IκB comprising at least one of Ser32 andSer 36, and a candidate agent; under conditions whereby, but for thepresence of said agent, said polypeptide specifically phosphorylatessaid IκB polypeptide at at least one of said Ser32 and Ser36 at areference activity; detecting the polypeptide-induced phosphorylation ofsaid IκB polypeptide at at least one of said Ser32 and Ser36 todetermine an agent-biased activity, wherein a difference between theagent-biased activity and the reference activity indicates that saidagent modulates the ability of said polypeptide to specificallyphosphorylate a IκB polypeptide.
 10. A method for modulating signaltransduction involving IκB in a cell, said method comprising the step ofmodulating IKK-α (SEQ ID NO:4) kinase activity.
 11. The method of claim10, wherein said modulating step comprises contacting the cell with aserine/threonine kinase inhibitor.